RESUMO
OBJECTIVES: In this study, we tested whether iodide would reduce heart damage in rat and pig models of acute myocardial infarction as a risk analysis for a human trial. DESIGN: Prospective blinded and randomized laboratory animal investigation. SETTING: Animal research laboratories. SUBJECTS: Sexually mature rats and pigs. INTERVENTIONS: Acute myocardial infarction was induced by temporary ligation of the coronary artery followed by reperfusion. Iodide was administered orally in rats or IV in rats and pigs just prior to reperfusion. MEASUREMENTS AND MAIN RESULTS: Damage was assessed by blood cardiac troponin and infarct size; heart function was determined by echocardiography. Blood peroxide scavenging activity was measured enzymatically, and blood thyroid hormone was determined using radioimmune assay. Iodide administration preserved heart function and reduced blood cardiac troponin and infarct size by approximately 45% in pigs and approximately 60% in rats. Iodide administration also increased blood peroxide scavenging activity and maintained thyroid hormone levels. CONCLUSIONS: Iodide administration improved the structure and function of the heart after acute myocardial infarction in rats and pigs.
Assuntos
Modelos Animais de Doenças , Iodetos/administração & dosagem , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Animais , Vasos Coronários/efeitos dos fármacos , Ecocardiografia , Estudos Prospectivos , Distribuição Aleatória , Ratos , Suínos , Glândula Tireoide/efeitos dos fármacosRESUMO
Chronic tissue ischemia due to defective vascular perfusion is a hallmark feature of peripheral artery disease for which minimal therapeutic options exist. We have reported that sodium nitrite therapy exerts cytoprotective effects against acute ischemia/reperfusion injury in both heart and liver, consistent with the model of bioactive NO formation from nitrite during ischemic stress. Here, we test the hypothesis that chronic sodium nitrite therapy can selectively augment angiogenic activity and tissue perfusion in the murine hind-limb ischemia model. Various therapeutic doses (8.25-3,300 mug/kg) of sodium nitrite or PBS were administered. Sodium nitrite significantly restored ischemic hind-limb blood flow in a time-dependent manner, with low-dose sodium nitrite being most effective. Nitrite therapy significantly increased ischemic limb vascular density and stimulated endothelial cell proliferation. Remarkably, the effects of sodium nitrite therapy were evident within 3 days of the ischemic insult demonstrating the potency and efficacy of chronic sodium nitrite therapy. Sodium nitrite therapy also increased ischemic tissue nitrite and NO metabolites compared to nonischemic limbs. Use of the NO scavenger carboxy PTIO completely abolished sodium nitrite-dependent ischemic tissue blood flow and angiogenic activity consistent with nitrite reduction to NO being the proangiogenic mechanism. These data demonstrate that chronic sodium nitrite therapy is a recently discovered therapeutic treatment for peripheral artery disease and critical limb ischemia.
Assuntos
Artérias/efeitos dos fármacos , Citoproteção , Membro Posterior/irrigação sanguínea , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Nitrito de Sódio/administração & dosagem , Animais , Artérias/crescimento & desenvolvimento , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Sequestradores de Radicais Livres/farmacologia , Imidazóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Doenças Vasculares Periféricas/tratamento farmacológico , Nitrito de Sódio/uso terapêuticoRESUMO
The HGF/c-Met pathway is an important regulator of signaling pathways responsible for invasion and metastasis of most human cancers, including prostate cancer. Exposure of DU145 prostate tumor cells to HGF stimulates the PI3-kinase and MAPK pathways, leading to increased scattering, motility, and invasion, which was prevented by the addition of EGCG. EGCG acted at the level of preventing phosphorylation of tyrosines 1234/1235 in the kinase domain of the c-Met receptor without effecting dimerization. HGF-induced changes were independent of the formation of reactive oxygen species, suggesting that EGCG functioned independent of its antioxidant ability. ECG, another tea polyphenol, was as effective as EGCG, while EGC and EC were less effective. EGCG added up to 4 h after the addition of HGF still blocked cell scattering and reduced the HGF-induced phosphorylation of c-Met, Akt, and Erk, suggesting that EGCG could act both by preventing activation of c-Met by HGF and by attenuating the activity of pathways already induced by HGF. HGF did not activate the MAPK and PI3-K pathways in cells treated with methyl-beta-cyclodextrin (mCD) to remove cholesterol. Furthermore, subcellular fractionation approaches demonstrated that only phosphorylated c-Met accumulated in Triton X-100 membrane insoluble fractions, supporting a role for lipid rafts in regulating c-Met signaling. Finally, EGCG treatment inhibited DiIC16 incorporation into membrane lipid ordered domains, and cholesterol partially inhibited the EGCG effects on signaling. Together, these results suggest that green tea polyphenols with the R1 galloyl group prevent activation of the c-Met receptor by altering the structure or function of lipid rafts.
Assuntos
Anticarcinógenos/farmacologia , Antioxidantes/farmacologia , Flavonoides/farmacologia , Catequina/análogos & derivados , Catequina/farmacologia , Movimento Celular/efeitos dos fármacos , Células/metabolismo , Humanos , Masculino , Octoxinol/metabolismo , Octoxinol/farmacologia , Fenóis/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol 3-Quinases/farmacologia , Fosforilação/efeitos dos fármacos , Polifenóis , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Chá , beta-CiclodextrinasRESUMO
Previous studies indicate that endothelial nitric oxide synthase (eNOS) function is impaired in diabetes as a result of increased vascular generation of reactive oxygen species. We hypothesized that eNOS gene therapy would augment NO. bioavailability and protect against hepatic ischemia-reperfusion (I-R) injury in type 2 diabetes mellitus. We developed a transgenic (Tg) diabetic mouse in which eNOS is systemically overexpressed. We also examined the effects of hepatic eNOS adenovirus therapy in diabetic mice. Diabetic (db/db) and nondiabetic mice were subjected to hepatic I-R injury. In nondiabetic mice, genetic overexpression of eNOS (both eNOS-Tg and eNOS adenovirus) resulted in hepatoprotection. In contrast, hepatic I-R injury was significantly increased in the db/db eNOS-Tg mouse, as serum alanine aminotransaminase (ALT) levels were increased by 3.3-fold compared with diabetic controls. Similarly, eNOS adenovirus treatment resulted in a 3.2-fold increase in serum ALT levels as compared with diabetic controls. We determined that hepatic eNOS was dysfunctional in the db/db mouse and increased genetic expression of eNOS resulted in greater production of peroxynitrite. Treatment with the eNOS cofactor tetrahydrobiopterin (BH4) or the BH4 precursor sepiapterin resulted in a significant decrease in serum ALT levels following I-R injury. We present clear examples of the protective and injurious nature of NO. therapy in I-R. Our data indicate that eNOS exists in an "uncoupled" state in the setting of diabetes and that "recoupling" of the eNOS enzyme with cofactor therapy is beneficial.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/terapia , Terapia Genética/efeitos adversos , Fígado/irrigação sanguínea , Óxido Nítrico Sintase Tipo III/genética , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Disponibilidade Biológica , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Biopterinas/farmacologia , Diabetes Mellitus Tipo 2/genética , Sinergismo Farmacológico , Fígado/metabolismo , Metaloporfirinas/farmacologia , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/farmacologia , Fenótipo , Fosforilação , Pterinas/farmacologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Índice de Gravidade de Doença , Tirosina/análogos & derivados , Tirosina/biossíntese , Tirosina/metabolismoRESUMO
BACKGROUND: Peripheral artery disease (PAD) is a prevalent cardiovascular disorder that results in tissue ischemia which can progress to critical limb ischemia. Restoration of tissue perfusion in the setting of chronic ischemia through stimulation of arteriogenesis and angiogenesis remains a key therapeutic target for PAD. However, experimental therapeutics, including growth factor and gene therapy, have had little clinical success indicating the need for a better understanding of molecular pathways required for therapeutic angiogenesis. METHODS AND RESULTS: Here we report that phosphodiesterase-5 inhibition by sildenafil significantly increases vascular perfusion, tissue blood flow, and vascular density during chronic ischemia of the mouse hind limb. Importantly, sildenafil therapy did not alter any of these parameters in nonischemic limbs. Sildenafil increased tissue cGMP levels independently of increases in nitric oxide production, and sildenafil therapy stimulated angiogenesis in ischemic limbs of eNOS-/- and iNOS-/- mice. Lastly, sildenafil-mediated angiogenic activity was blocked by inhibition of protein kinase G using the PKG antagonist DT-3. CONCLUSIONS: These data demonstrate that sildenafil therapy results in increased angiogenic activity through a PKG-dependent pathway that is independent of nitric oxide production or NOS activity and identify the angiogenic therapeutic potential of sildenafil for critical limb ischemia.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Isquemia/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Piperazinas/farmacologia , Sulfonas/farmacologia , Animais , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Membro Posterior/irrigação sanguínea , Isquemia/fisiopatologia , Masculino , Camundongos , Doenças Vasculares Periféricas/tratamento farmacológico , Purinas/farmacologia , Fluxo Sanguíneo Regional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Citrato de SildenafilaRESUMO
Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1(-/-) mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1(-/-) mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1(-/-) MAEC to levels observed in WT MAEC with 150 microM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1(-/-) cells. Deficient angiogenic responses in ICAM-1(-/-) cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.
Assuntos
Glutationa/metabolismo , Molécula 1 de Adesão Intercelular/fisiologia , Neovascularização Fisiológica , Óxido Nítrico Sintase Tipo II/metabolismo , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Aorta/metabolismo , Movimento Celular , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Óxido Nítrico Sintase Tipo III , PTEN Fosfo-Hidrolase/metabolismoRESUMO
What mechanisms regulate endothelial glutathione (GSH) during inflammation? Addressing this question is critical in understanding mechanisms leading to endothelial dysfunction and cardiovascular disease. Herein, the authors show data that support the hypothesis that the intercellular cell adhesion molecule-1 (ICAM-1) regulates GSH. Ligating either constitutive or induced ICAM-1 on the endothelial surface, or exposing endothelial cells to soluble ICAM-1, increases GSH concentrations. ICAM-1 is important in mediating leukocyte adhesion and modulates endothelial signaling pathways important in controlling transmigration. The present data underscore a novel function for ICAM-1 in modulating GSH metabolism and raise the hypothesis that this adhesion molecule controls endothelial redox status under basal and inflammatory conditions.
Assuntos
Reagentes de Ligações Cruzadas , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Glutationa/metabolismo , Molécula 1 de Adesão Intercelular/fisiologia , Animais , Células Cultivadas , Células Endoteliais/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Camundongos , Camundongos Knockout , OxirreduçãoRESUMO
OBJECTIVE: The protective effect of NO synthase-3 (eNOS)-derived NO in limiting myocardial ischemia-reperfusion (MI-R) injury is well established. We reported previously that systemic genetic overexpression of eNOS attenuates MI-R injury. The purpose of the current study was to investigate tissue-specific genetic overexpression of the human eNOS gene. METHODS AND RESULTS: To accomplish this, we used 2 distinct murine models of transgenic overexpression, a cardiomyocyte-specific eNOS overexpresser (CS eNOS-Tg) under the control of the alpha-myosin heavy chain promoter, and a systemic eNOS transgenic mouse (SYS eNOS-Tg) under control of the native eNOS promoter with an upstream endothelial enhancer element. Mice were subjected to 30 or 45 minutes of left coronary artery ischemia and 24 or 72 hours of reperfusion. CS eNOS-Tg mice displayed significantly decreased infarct size beyond that of mice with systemic overexpression. Additionally, CS eNOS-Tg mice exhibited better preservation of cardiac function compared with SYS eNOS-Tg mice after myocardial infarction. CONCLUSIONS: These results provide evidence that site-specific targeting of eNOS gene therapy may be more advantageous in limiting MI-R injury and subsequent cardiac dysfunction.
Assuntos
Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Ecocardiografia , Marcação de Genes , Coração/fisiopatologia , Hemodinâmica , Humanos , Camundongos , Camundongos Transgênicos , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/enzimologia , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Distribuição TecidualRESUMO
We previously reported that ICAM-1 expression modulates endothelial intracellular glutathione (GSH) metabolism through unknown mechanisms. Here we report that the cytoplasmic tail of ICAM-1 is critically involved in governing intracellular GSH production. Peptides containing the antennapedia cell-permeative sequence (AP) or an AP peptide linked to the transmembrane and cytosolic tail of ICAM-1 (AP-ICAM) were synthesized and used to measure alterations in redox status in cultured endothelial cells and determine their biological effect. Treatment with AP-ICAM significantly increased GSH concentrations and glutamate-cysteine ligase (GCL) activity over time. Measuring reactive oxygen species (ROS) production with DCF revealed a rapid increase in ROS generation after AP-ICAM treatment. Measurement of superoxide production with hydroethidium revealed biphasic production at 30 min and 6h after treatment with AP-ICAM. Apocynin, DPI, catalase, or SOD attenuated AP-ICAM-dependent ROS production, GCL activity, and GSH production, implicating superoxide production and dismutation to peroxide. Consistent with these findings, NOX4 siRNA knockdown blocked AP-ICAM peptide increases in GSH or GCL activity, demonstrating the importance of NADPH oxidase. Last, inhibition of PI3-kinase activity with LY 294002 or wortmannin blocked AP-ICAM GSH induction and ROS production. These data reveal that the ICAM-1 cytoplasmic tail regulates production of endothelial GSH through a NOX4/PI3-kinase-dependent redox-sensitive pathway.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Glutamato-Cisteína Ligase/metabolismo , Molécula 1 de Adesão Intercelular/farmacologia , NADPH Oxidases/metabolismo , Fragmentos de Peptídeos/farmacologia , Acetofenonas/farmacologia , Androstadienos/farmacologia , Animais , Linhagem Celular , Cromonas/farmacologia , Citosol/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Ativação Enzimática/efeitos dos fármacos , Glutamato-Cisteína Ligase/genética , Glutationa/biossíntese , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Morfolinas/farmacologia , NADPH Oxidase 4 , NADPH Oxidases/genética , Fragmentos de Peptídeos/síntese química , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de Proteína/genética , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , WortmaninaRESUMO
Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders of the intestinal tract with unknown multifactorial etiology that, among other things, result in alteration and dysfunction of the intestinal microvasculature. Clinical observations of increased colon microvascular density during IBD have been made. However, there have been no reports investigating the physiological or pathological importance of angiogenic stimulation during the development of intestinal inflammation. Here we report that the dextran sodium sulfate and CD4+CD45RBhigh T-cell transfer models of colitis stimulate angiogenesis that results in increased blood vessel density concomitant with increased histopathology, suggesting that the neovasculature contributes to tissue damage during colitis. We also show that leukocyte infiltration is an obligatory requirement for the stimulation of angiogenesis. The angiogenic response during experimental colitis was differentially regulated in that the production of various angiogenic mediators was diverse between the two models with only a small group of molecules being similarly controlled. Importantly, treatment with the anti-angiogenic agent thalidomide or ATN-161 significantly reduced angiogenic activity and associated tissue histopathology during experimental colitis. Our findings identify a direct pathological link between angiogenesis and the development of experimental colitis, representing a novel therapeutic target for IBD.
Assuntos
Vasos Sanguíneos/fisiopatologia , Colite/induzido quimicamente , Colo/irrigação sanguínea , Neovascularização Patológica/etiologia , Talidomida/farmacologia , Inibidores da Angiogênese/farmacologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Colite/patologia , Colo/patologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de NeutrófilosRESUMO
Leukocyte recruitment into pancreatic islets is believed to play an important pathophysiological role in autoimmune diabetes. Previous reports have suggested that several different adhesion molecules may be involved in leukocyte recruitment during autoimmune diabetes, including members of the leukocyte beta(2) integrins. Here we report that a gene-targeted deficiency of the beta(2) integrin, CD18, protects against multiple low-dose streptozotocin-induced autoimmune diabetes. CD18 null mice displayed lower blood glucose values throughout the study, with only 10% of these mice eventually developing diabetes compared to 95% in the control group. Importantly, the development of insulitis was markedly absent in the CD18 null mice, suggesting that members of this integrin subfamily predominately regulate leukocyte infiltration into pancreatic islets. This study demonstrates that the beta(2) integrins play a key pathophysiological role in the development of multiple low-dose streptozotocin-induced autoimmune diabetes.
Assuntos
Antígenos CD18/fisiologia , Diabetes Mellitus Experimental/prevenção & controle , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/etiologia , Relação Dose-Resposta a Droga , Marcação de Genes , Hiperglicemia/patologia , Ilhotas Pancreáticas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Coordinated regulation of endothelial cell migration is an integral process during angiogenesis. However, molecular mechanisms regulating endothelial cell migration remain largely unknown. Increased expression of cell adhesion molecules has been implicated during angiogenesis, yet the precise role of these molecules is unclear. Here, we examined the hypothesis that intercellular adhesion molecule-1 (ICAM-1) is important for endothelial cell migration. Total cell displacement and directional migration were significantly attenuated in ICAM-1-deficient endothelium. Closer examination of ICAM-1-deficient cells revealed decreased Akt Thr(308) and endothelial nitric-oxide synthase Ser(1177) phosphorylation and NO bioavailability, increased actin stress fiber formation, and a lack of distinct cell polarity compared with wild-type endothelium. Supplementation of ICAM-1 mutant cells with the NO donor DETA NONOate (0.1 microM) corrected the migration defect, diminished stress fiber formation, and enhanced pseudopod and uropod formation. These data demonstrate that ICAM-1 facilitates the development of cell polarity and modulates endothelial cell migration through a pathway regulating endothelial nitric-oxide synthase activation and organization of the actin cytoskeleton.